*4.7. Anti-Viral*

Several phytochemicals isolated from various Artemisia species exhibit significant antiviral activity [76]. ARTs have turned out to be the most promising antiviral drug candidates with activities against hepatitis B and C viruses, human herpes viruses HSV-1 and HSV-2, HIV-1 and influenza virus A in the low micromolar range [77–82]. In most cases, ARTs inhibited the central regulatory processes of viral-infected cells (NF-κB or Sp1-dependent pathways), thus blocking the host-cell–type and metabolic requirements for viral replication [80].

Owing to their potent anti-inflammatory, immunoregulatory and antiviral properties, ARTs are being pursued for their activity against SARS-CoV-2 infection. Researchers used in silico approaches to investigate if artemisinin or its derivatives could physically bind any of the COVID-19 target proteins including SARS-CoV-2 spike glycoprotein, spike ectodomain structural protein, the main protease of the virus (MPro) or spike receptor-binding domain, thereby preventing SARS-CoV-2 from binding to the host receptor ACE2 [83–89]. AD-MET (absorption, distribution, metabolism, excretion and toxicity) analysis of artemisinin showed that it was non-cytotoxic, had good aqueous solubility and a good permeability through the blood–brain barrier with a promising therapeutic potential. Furthermore, molecular docking studies revealed that artemisinin bound to all four proteins and in some cases displayed better binding modes than hydroxychloroquine [85–89]. Thus, ARTs could serve as best leads for further drug development process for SARS-CoV-2 infection.

Several investigators have now shown that extracts from different species of Artemisia are active against SARS-CoV-2 [79,86,90,91]. Results from recent studies indicate that ARTs impair SARS-CoV-2 viral infection by modulating several host cell metabolic pathways thus making them attractive candidates for COVID-19 [85,86,92]. The mechanism of antiviral activity may be through the induction of cellular ROS, blunting the PI3K/Akt/p70S6K signaling pathway, binding to NF-κB/Sp1 or inducing a endocytosis inhibition mechanism, all of which lead to inhibition of viral replication and growth [85,93,94]. The above mentioned results have spurred the interest of few groups to embark on clinical trials to evaluate the safety and efficacy of ARTs in the treatment of subjects with SARS-CoV-2 viral infection.

In a recently published controlled clinical trial, 41 patients with confirmed COVID-19 were divided into two groups. While 18 subjects served as the control group, the experimental group (*n* = 23) received a combination of artemisinin-piperaquine (AP). AP was orally administrated with a loading dose of two tablets (artemisinin 125 mg and piperaquine 750 mg) on the first day, followed by a low dose of one tablet/day (artemisinin 62.5 mg and piperaquine 375 mg) for six days [95]. The primary outcome was the percentage of participants with undetectable SARS-CoV-2 on days 7, 10, 14, and 28 following the treatment. The results indicated that: (1) the average time to achieve undetectable SARS-CoV-2 RNA in the AP group was significantly less than that in the control group; (2) the elimination rate of SARS-CoV-2 RNA in the AP group was significantly higher than that in the control group; and (3) the length of hospital stay for the AP group was significantly lower than that in the control group. Although the study had insufficient sample size and trial design, nevertheless, the safe toxicity profile and immunoregulatory activities makes AP an excellent drug candidate against SARS-CoV-2 infection [95].

Transforming Growth Factor-beta (TGF-β) plays an important role in modulating the immune system and displays different activities on different types of immune cells. SARS- CoV-2 infection is accompanied by a cytokine storm together with edema and pulmonary fibrosis at the end stage of the infection. SARS-CoV-2 also up-regulates TGF-β expression which may partly explain the cytokine storm and fibrosis in the lung [94,96,97]. Efforts are underway to discover novel and specific small molecules that can potently block TGF-β expression with negligible side-effects. Artemisinin and its derivatives have been shown to be suppressors of TGF-β in several models of inflammatory diseases [64,98–100]. A randomized, open-label Phase IV study is underway to evaluate the safety and efficacy of a proprietary formulation of ARTs in adult COVID-19 patients with symptomatic mildmoderate COVID-19 [101]. In addition to its potent antiviral activity, the drug is expected to mitigate the TGF-β mediated inflammatory injury associated with the cytokine storm and viral sepsis in these patients. Initial results show that the ARTs-based drug has a very favorable safety profile and significantly accelerated the recovery of patients with mild-moderate COVID-19 infection [101]. Thus inhibition of TGF-β signaling by ARTs may be an attractive therapeutic strategy making them excellent drug candidates against SARS-CoV-2 infection.

#### **5. Conclusions and Future Direction**

Several phytochemical derivatives and lead molecules have been developed from medicinal plants for various significant therapeutic activities [4,5,94,102,103]. Scientists routinely investigate medicinal plants just for that one single and potent compound responsible for the therapeutic effect [104]. Studies comparing the action of whole plant extracts to the action of purified preparation show that, in many cases, the potency of the purified preparation declines at each step of fractionation [105]. Since the therapeutic effect may be the result of the combination of several compounds present in the medicinal plant, a complex mixture of compounds has a greater effect than isolated compounds [106]. The advantages of a combinatorial approach may be the synergy exhibited by the various components, enhanced bioavailability, cumulative effects and affecting an entire network of pathways in tandem [107].

Artemisia has prominence in Chinese and Ayurvedic medicinal systems for its numerous therapeutic properties. Among the phytochemicals present in the plant, the lactone derivative artemisinin and its derivatives—termed ARTs—are very promising owing to their multiple pharmacological actions [4,7,85,93,94]. Recent studies point to ARTs as attractive candidates for SARS-CoV-2 and they are a major focus in medical research [4,85,92,103]. SARS-CoV-2 infection manifests as a mild respiratory tract infection and influenza-like illness to a severe disease with accompanying lung injury (in severe cases lung fibrosis), oxidative stress, multisystem inflammatory conditions, multi-organ failure and neurological issues [108–111]. The role of ARTs as an antioxidant and anti-inflammatory and to be able to block tissues fibrosis together with its safety and low toxicity profile makes it an excellent drug candidate against SARS-CoV-2 infection [85,94].

**Author Contributions:** Conceptualization, S.G.K. and R.V.R.; Methodology, S.G.K. and R.V.R.; Data curation, S.G.K. and R.V.R.; Original draft preparation and Writing, R.V.R.; Reviewing and Editing, S.G.K. and R.V.R. Both authors have read and agreed to the published version of the manuscript.

**Funding:** The research study did not receive any specific gran<sup>t</sup> from funding agencies in the public, commercial, or not-for-profit sectors.

**Institutional Review Board Statement:** Not Applicable.

**Informed Consent Statement:** Not Applicable.

**Data Availability Statement:** Not Applicable.

**Conflicts of Interest:** This manuscript is not under consideration by another journal, nor has it been published. SGK is a paid consultant-at-large with Oncotelic, Inc., a wholly-owned subsidiary of Mateon Therapeutics, the sponsor for the clinical development of the proprietary formulation of ARTs for SARS-CoV-2 infection. RVR declares no competing financial interests.
